Methods and compositions for producing biopolymeric arrays

ABSTRACT

Methods and compositions are provided for producing arrays of polymeric binding agents. In the subject methods, the individual polymers of the array are synthesized using solid phase synthesis techniques on the surface of a substrate. A critical feature of the invention is that one or more locations on the substrate surface are spatially and temporally protected by a protective bubble during the synthesis protocol, where the protective bubble may be produced using any convenient bubble producing means. The bubble producing means may be a component of either a substrate or a structure separate from the substrate. Also provided are the arrays produced by the subject methods, kits for use in practicing the subject methods, and methods of using the arrays in analyte detection assays, including hybridization assays, such as gene discovery, differential gene expression and gene sequencing assays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/997,564, filed Nov. 28, 2001, which is a divisional of Ser. No.09/354,816, filed Jul. 16, 1999, now issued U.S. Pat. No. 6,346,423 fromwhich priority is claimed under 35 U.S.C. 120. The entireties of theseapplications are incorporated herein by reference.

INTRODUCTION

1. Technical Field

The field of this invention is polymeric arrays.

2. Background of the Invention

“Biochips” or arrays of binding agents, such as oligonucleotides andpeptides, have become an increasingly important tool in thebiotechnology industry and related fields. These binding agent arrays,in which a plurality of binding agents are present on a solid supportsurface in the form of an array or pattern, find use in a variety ofapplications, including gene expression analysis, drug screening,nucleic acid sequencing, mutation analysis, and the like.

Such arrays may be prepared in a number of different ways. For example,DNA arrays may be prepared manually by spotting DNA onto the surface ofa substrate with a micro pipette. See Khrapko et al., DNA Sequence(1991) 1:375-388. Alternatively, the dot-blot approach, as well as thederivative slot-blot approach, may be employed in which a vacuummanifold transfers aqueous DNA samples from a plurality of wells to asubstrate surface. In yet another method of producing arrays ofbiopolymeric molecules, a pin is dipped into a fluid sample of thebiopolymeric compound and then contacted with the substrate surface. Byusing a plurality or array of pins, one can transfer a plurality ofsamples to the substrate surface at the same time. Alternatively, anarray of capillaries can be used to produce biopolymeric arrays. See WO95/35505. In another method of producing biopolymeric arrays, arrays ofbiopolymeric agents are “grown” on the surface of a substrate indiscreet regions. See e.g. U.S. Pat. No. 5,143,854 and Fodor et al.,Science (1991) 251:767-773.

Despite the variety of different methods available for the production ofbiopolymeric arrays, there are disadvantages associated with eachmethod. For example, current methods of growing the polymeric agents onthe surface of an array, such as the photoresist techniques described inFodor supra, are expensive and require the use of specializedphotosensitive protecting groups on the phosphoramidites. As such, thereis continued interest in the development of new methods for producingpolymeric arrays, particularly in the development of new methods forgrowing polymers on the surface of a substrate to produce an array.

Relevant Literature

Patents and patent applications describing arrays of biopolymericcompounds and methods for their fabrication and/or use include: U.S.Pat. Nos. 4,877,745; 5,143,854; 5,242,974; 5,338,688; 5,384,261;5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327;5,445,934;5,449,754; 5,472,672; 5,474,796; 5,510,270; 5,527,681;5,529,756; 5,532,128; 5,545,531; 5,552,270; 5,554,501; 5,556,752;5,658,802; 5,561,071; 5,599,695; 5,624,711; 5,639,603; 5,658,734;5,670,322; 5,677,195; 5,698,089; 5,700,637; 5,723,320; 5,744,305;5,759,779; 5,763,170; 5,846,708; WO 90/10716; WO 92/10588; WO 93/17126;WO 95/11995; WO 95/35505; WO 97/10365; WO 97/27317; WO 97/46313; EP 0373 203 B1; EP 742 287 A2; and EP 799 897 A1.

SUMMARY OF THE INVENTION

Methods and compositions for making polymeric arrays are provided. Inthe subject methods, polymers are produced through the sequentialcovalent addition of polymeric subunits to a growing polymer chain onthe surface of a substrate, where one or more locations of the substratesurface are selective protected (both spatially and temporally) by aprotective bubble during the sequential synthesis protocol. Theprotective bubble may be produced on the surface in any convenientmanner, including through activation of bubble producing means, e.g.resistors, stably associated with a surface of the substrate or part ofa structure separate from the substrate. A variety of different types ofpolymeric arrays can be produced according to the subject methods,including polypeptide and nucleic acid arrays. The subject arrays finduse in a variety of different analyte detection applications, includinghybridization assays, where specific applications include genediscovery, differential expression and nucleic acid sequencing assays.

Definitions

The term “nucleic acid” as used herein means a polymer composed ofnucleotides, e.g. deoxyribonucleotides or ribonucleotides.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “oligonucleotide” as used herein denotes single strandednucleotide multimers of from about 10 to up to about 100 nucleotides inlength.

The term “polynucleotide” as used herein refers to a single or doublestranded polymer composed of nucleotide monomers of generally greaterthan 100 nucleotides in length and up to about 8,000 or more nucleotidesin length.

The term “peptide” as used herein refers to any compound produced byamide formation between a carboxyl group of one amino acid and an aminogroup of another group.

The term “oligopeptide” as used herein refers to peptides with fewerthan about 10 to 20 residues, i.e. amino acid monomeric units.

The term “polypeptide” as used herein refers to peptides with more than10 to 20 residues.

The term “protein” as used herein refers to polypeptides of specificsequence of more than about 50 residues.

The term “array” as used herein means an substrate having a plurality ofbinding agents stably attached to its surface, where the binding agentsmay be spatially located across the surface of the substrate in any of anumber of different patterns.

The term “binding agent” means any agent that is a member of a specificbinding pair, where such agents include: peptides, e.g. proteins orfragments thereof; nucleic acids, e.g. oligonucleotides,polynucleotides; and the like; etc.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for producing arrays of polymericbinding agents. In the subject methods, the individual polymers of thearray are synthesized using solid phase synthesis techniques on thesurface of a substrate. A critical feature of the invention is that oneor more locations on the substrate surface are spatially and temporallyprotected by a protective bubble during the synthesis protocol, wherethe protective bubble may be produced using any convenient bubbleproducing means. The bubble producing means may be a component of eithera substrate or a structure separate from the substrate. Also providedare the arrays produced by the subject methods, kits for use inpracticing the subject methods, and methods of using the arrays inanalyte detection assays, including hybridization assays, such as genediscovery, differential gene expression and gene sequencing assays.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

The subject invention provides methods for fabricating arrays ofpolymeric agents. The subject invention can be used to fabricate anumber of different types of arrays in which a plurality of distinctpolymeric binding agents are stably associated with at least one surfaceof a substrate. The polymeric binding agents may vary widely, where theonly limitation is that the polymeric binding agents are capable ofbeing fabricated in a step-wise fashion in which sub-units of thepolymer, e.g. monomeric units, submonomers, macromonomers (i.e.compounds of two or more, usually no more than 10 and more usually nomore than 8 monomers), are sequentially added to each other to form agrowing polymeric chain to ultimately produce a polymeric molecule.Polymeric binding agents of particular interest include biopolymericmolecules, such as peptides, nucleic acids, polysaccharides and thelike, where peptides and nucleic acids are of particular interest inmany embodiments.

A critical feature of the subject methods is that protective bubbles arepositioned on the surface of the substrate both spatially and temporallyduring the synthesis protocol in order to control the nature (i.e.sequence) of the polymers that are produced on the substrate surface. Byspatially is meant that the location of the protective bubble(s) isselected in a specific manner, such that the protective bubble protectsdiscrete and defined locations on the support surface during polymersynthesis. By temporally is meant that the duration and life of theprotective bubble is selected such that a location on the substratesurface may be protected at one stage during synthesis and not protectedat a second stage during synthesis. By modulating the location of theprotective bubbles on the surface of the substrate during synthesis,arrays of polymers of defined sequence and location are produced.

The substrate surface may be protected with protective bubbles using anyconvenient means. Generally, a protective bubble producing means isemployed to produce the protective bubble(s) that selectively protectone or more locations of the substrate surface at a given time duringpolymer synthesis. Although any type of bubble producing means that iscapable of being activated to produce a bubble in a solvent layerpositioned above the surface of the substrate during polymer synthesismay be employed, the bubble producing means is generally a heatingmeans. In many embodiments, the heating means is a resistor. Forconvenience, the subject invention is now further described in terms ofthese embodiments in which the bubble producing means is a resistor.

The protective bubble producing means employed to produce the protectivebubble(s) may incorporated into either the substrate or a structureseparate from the substrate, with the only requirement being that thebubble producing means must be capable of protecting the substratesurface at the desired location and time during the synthesis process.Thus, in a first embodiment of the invention, the bubble producing meansis a component of the substrate on which the polymers are grown, e.g.resistors are components of the substrate, as described in greaterdetail below. In a second embodiment, the bubble producing means (e.g.resistor) is a component of a structure separate from the substrate,such as a second plate that is brought into sufficient proximity of thesubstrate surface during synthesis, as described in greater detailbelow.

The substrate employed in the subject invention may be any convenientconfiguration, but generally has a planar configuration. By “planarconfiguration” is meant that the substrate has at least one planarsurface, which surface may have any convenient cross-sectional shape,including circular, oval, square, rectangular and the like. In manyembodiments, the substrate has a plate-like configuration, such as isfound in a disk, rectangular slide, square slide, and the like. In manyembodiments in which the ultimate array is to have a planarconfiguration, the substrate comprises at least one planar surface thathas a cross-sectional area of at least about 4 mm³, usually at leastabout 16 mm² and more usually at least about 25 mm², where thecross-sectional area of the planar surface may be as large as 2500 mm²or larger, but generally does not exceed about 900 mm² and usually doesnot exceed about 400 mm². In those embodiments where the planar surfacehas a square or rectangular shape, the planar surface has a length offrom about 2 to 50 mm, usually from about 4 to 30 mm and more usuallyfrom about 5 to 20 mm, and has a width ranging from about 2 to 50 mm,usually from about 4 to 30 mm and more usually from about 5 to 20 mm.The substrate thickness may vary considerably, depending on thedetection protocol, i.e. whether detection is through the substrate orjust on the surface. For example, where the array is to be read throughthe substrate, the thickness generally ranges from about 0.7 to 1.2 mm.Alternatively, where the array is to be surface read, the thickness isgenerally dictated by the substrate fabrication process.

In the first embodiment of the subject invention mentioned above, thesubstrate employed in the subject methods is a substrate that has aplurality of distinct, individually controllable resistors associatedwith at least one of its surfaces, i.e. the surface on which it isdesired to place one or more polymeric compounds. More particularly, thesubstrate in this first embodiment is generally made up of a basematerial having a plurality of resistors positioned on at least onesurface thereof and then coated with an insulating layer that ismodified, as necessary, to provide for any surface chemistry requisitefor production of the polymer molecules on the substrate surface. Thebase material of the substrate is fabricated from any convenientmaterial or materials, where the substrate may be a pure material or acomposite structure of two or more different materials. As indicatedabove, the substrate can be fabricated from any material on which aplurality of resistors can be surface mounted. Suitable materialstherefore include silicon, fused glass, silica, and the like. Ideally,the substrate will have a surface that provided at most low backgroundfluorescence.

On at least one surface of the base material, generally the planarsurface of the base material, are a plurality of individuallyactivatable resistors. By plurality is meant at least 2, usually atleast 10 and more usually at least 100, where the number of resistorspresent on the substrate surface may be as high as 100,000 or higher,but generally does not exceed about 20,000 and usually does not exceedabout 10,000.

The resistors are generally positioned on the planar surface of the basematerial in the form of a pattern. Where the resistors are positioned onthe base material in the form of a pattern, the pattern may vary asdesired. As such, the pattern may be in the form of organized rows andcolumns of resistors, e.g. a grid of resistors, across the base materialsurface, a series of curvilinear rows across the substrate surface, e.g.a series of concentric circles or semi-circles of resistors, and thelike.

The spacing of the resistors on the base material surface is sufficientto provide a density of at least about 1000/cm², usually at least about2000/cm² and more usually at least about 2500/cm², where the density maybe as great as 40,000/cm² or greater, but generally does not exceedabout 10,000/cm² and usually does not exceed about 4,000/cm². In manyembodiments the distance between any two given resistors on a basematerial surface is at least about 50 μm, usually at least about 100 μmand more usually at least about 160 μm, where the distance between anytwo adjacent resistors on the base material surface may be as great as 1mm or greater, but generally does not exceed about 0.5 mm and usuallydoes not exceed about 0.3 mm.

The resistors may be fabricated from any convenient material that iscapable of undergoing a sufficient rise in temperature followingapplication of an appropriate electrical current. As such, any materialthat is capable of achieving a temperature of at least about 50° C.,usually at least about 90° C. and more usually at least about 110° C.upon application of an electrical current having a magnitude of fromabout 1 to 100 mA, usually from about 10 to 50 mA, may be employed.Suitable materials from which the resistors may be fabricated include:tantalum nitride, tantalum aluminum alloy, and the like.

Each of the resistors is individually activatable. By individuallyactivatable is meant that the temperature of each resistor may be raisedor lowered separately from any other resistor present on the substrate.As such, means for providing the requisite electrical current to eachresistor are present on the base material surface, where such means maybe any current communication means, such as stripes of a conductingmaterial, etc. The means for applying electrical current to eachresistor further includes a means for controlling which resistors areactivated and which are not, i.e. a switching means for directingelectrical current to the appropriate resistors associated with thesubstrate surface. A variety of suitable means as described above areknown in the integrated circuit art and may be employed.

Positioned over the surface of the resistors on the base material is aninsulating material, i.e. an insulator layer. The insulator material maybe any convenient, non-conductive material or a composite of two or moreinert materials, where suitable inert materials include silicon dioxide,silicon nitride, silicon carbide and the like, with silicon dioxidebeing preferred, with the only proviso being that the surface of theinsulator layer must be susceptible to modification such that attachmentof polymers to the surface can be achieved during polymer synthesis. Thethickness of the insulator layer is typically at least about 0.1 andmore usually at least about 0.2 μm, where the insulator layer may have athickness as great as 2.0 μm or greater, but will generally not exceedabout 1.5 μm and usually will not exceed about 1.0 μm. As such, theplurality of the resistors is associated with the substrate surface, andmore specifically is beneath the substrate surface, i.e. underneath theinsulating layer of the substrate.

The surface of the insulator layer is generally modified to provide forsurface groups that allow for covalent attachment of the polymericsubunits onto the insulator layer during fabrication of the arrays.Surface reactive groups that may be introduced onto the surface of theinsulating layer include: amino, e.g. primary amino; hydroxyl; thiol;sulfonic acid; phosphorous and phosphoric acid, particularly in the formof acid halides; especially chloride and bromide; and the like. Thereactive groups may introduced onto the surface of the substrate usingany convenient protocol, such as the protocols described in U.S. patentapplication Ser. No. 09/145,015 filed Sep. 1, 1998, the disclosure ofwhich is herein incorporated by reference.

The above substrates may be fabricated using any convenient protocol,where standard IC fabrication and surface chemistry modificationprotocols are generally employed. A representative protocol is asfollows is provided in the experimental section infra.

In the second embodiment, the substrate is not limited as in the firstembodiment, in that the substrate employed in the second embodiment doesnot include a plurality of resistors. Instead, the bubble producingmeans, e.g. resistors, are present on a structure separate from thesubstrate, as summarized above. As such, the substrate may be anyconvenient substrate that finds use in biopolymeric arrays. In general,the substrate is rigid substrate in this second embodiment. By rigid ismeant that the support is solid and does not readily bend, i.e. thesupport is not flexible. As such, rigid substrates are sufficient toprovide physical support and structure to the nucleic acid spots presentthereon. Furthermore, when the rigid supports of the subject inventionare bent, they are prone to breakage. The substrates may be fabricatedfrom a variety of materials. In certain embodiments, e.g. where one isinterested in the production of nucleic acid arrays for use in researchand related applications, the materials from which the substrate may befabricated should ideally exhibit a low level of non-specific bindingduring hybridization events. In many situations, it will also bepreferable to employ a material that is transparent to visible and/or UVlight. For rigid substrates, specific materials of interest include:silicon; glass; plastics, e.g. polytetrafluoroethylene, polypropylene,polystyrene, polycarbonate, and blends thereof, and the like; metals,e.g. gold, platinum, and the like; etc. The surface may be modified withone or more different layers of compounds that serve to modify theproperties of the surface in a desirable manner. Such modificationlayers, when present, will generally range in thickness from amonomolecular thickness to about 1 mm, usually from a monomolecularthickness to about 0.1 mm and more usually from a monomolecularthickness to about 0.001 mm. Modification layers of interest include:inorganic and organic layers such as metals, metal oxides, conformalsilica or glass coatings, polymers, small organic molecules and thelike. Polymeric layers of interest include layers of: peptides,proteins, polynucleic acids or mimetics thereof, e.g. peptide nucleicacids and the like; polysaccharides, phospholipids, polyurethanes,polyesters, polycarbonates, polyureas, polyamides, polyethyleneamines,polyarylene sulfides, polysiloxanes, polyimides, polyacetates, and thelike, where the polymers may be hetero- or homopolymeric, and may or maynot have separate functional moieties attached thereto, e.g. conjugated.The particular surface chemistry will be dictated, as in the firstembodiment, by the specific process to be used in polymer synthesis, asdescribed in greater detail infra.

Turning now to the subject methods for producing arrays, the first stepof the subject methods is to produce a solvent layer on at least onesurface of the array, i.e. the surface on which the polymers are to besynthesized. The solvent layer may be produced on the surface of thesubstrate by any convenient protocol, such as by immersing the substratein a container of the solvent, flooding the surface of the array withthe solvent and the like. As such, the thickness of the solvent layermay vary widely, as long as it is sufficiently thick to provide for asuitable environment for the polymeric production chemistry to occur onthe substrate surface coated with the solvent layer. In other words, thesolvent layer must be of sufficient thickness to provide a suitablefluid environment for the polymeric synthesis chemical reactions tooccur. Where the surface of the substrate is flooded with the solventlayer, the thickness of the solvent layer is generally at least about100 μm, usually at least about 1 mm thick, where the thickness may be asgreat as 3 mm or greater, but generally does not exceed about 2.5 mm andusually does not exceed about 2 mm.

The solvent of the solvent layer is generally selected based on theparticular chemistry to be performed in production of the polymericbinding agents. As such, depending on the particular chemical reactionsto be performed during the synthesis of the polymers on the substratesurface, the solvent layer may be aqueous or non-aqueous. For example,the solvent layer may be aqueous in those situations where the solidphase polymeric synthesis protocols do not require anhydrous conditions.Aqueous solvents of interest include pure water, a water in combinationwith a co-solvent, e.g. an organic co-solvent, and the like. Whereanhydrous conditions are required, e.g. in nucleic acid synthesis,non-aqueous solvents are employed. Non-aqueous solvents of interestinclude organic solvents, such as tetrahydrofuran, acetonitrile,dichloromethane, and the like, where acetonitrile is preferred in manyembodiments, e.g. in the production of nucleic acid arrays.

Following production of the solvent layer on the substrate surface, thesubstrate surface is selectively protected. By “selectively protected”is meant that a portion of, but not all of, the potential reactive siteson the substrate surface (i.e. the sites containing covalently boundsusceptible reactive moieties) are protected from reaction. As such,following selective protection of the substrate surface, only a portionof the potential susceptible reactive groups present on the substratesurface are actually available for reaction with a correspondingreactive group, i.e. a group that reacts with the surface boundsusceptible moiety.

The substrate surface is selectively protected with one or moreprotective bubbles positioned on the surface at sites where preventionof reaction between surface bound susceptible groups and reactive agentsin the solvent layer is desired. The protective gas bubbles are producedat a desired site(s) on the substrate surface to achieve selectiveprotection of the substrate surface in any convenient manner.

In the first embodiment of the subject invention (i.e. where thesubstrate comprises a plurality of individually activatable resistors),selective protection of one or more sites is achieved by activating theresistor associated with the substrate surface at each site whereprotection is desired, i.e. by selectively activating the resistorsassociated with the substrate surface. In selectively activating theresistors associated with the substrate surface, only a portion of thetotal number of resistors is activated, where the portion that areactivated are those that underlie the surface at the site at whichprotection is desired.

In the second embodiment of the subject invention, as summarized abovethe resistors (or other bubble producing means) are present on astructure that is separate from the substrate. Typically, though notnecessarily, the resistors are present on a plate like structure ofanalogous proportions and composition to the substrate of the firstembodiment (with the exception that the surface need not be modified toprovide for functional groups employed in the polymer synthesisprotocol), such that the resistors can be lined up with discrete andknown locations of the substrate to protect these locations at variousstages during the synthesis. In these embodiments, the structurecomprising the resistors is brought within sufficiently close proximityto the substrate surface such that the bubble which nucleates on theseparate structure is capable of touching and covering a reactive siteon the substrate.

The resistors (present on either the substrate or the separatestructure, as described above) are selectively activated by applying anelectrical current to those resistors located at the site of either thesubstrate surface to be protected or on the separate structureimmediately opposite the substrate surface location to be protected. Thesurface area of the substrate covered by each bubble, i.e. the surfacearea of the substrate at which solvent is prevented from having contactwith the susceptible moieties stably attached thereto, generally rangesfrom about 100 to 62,500 μm², usually from about 625 to 22500 μm², andmore usually from about 2500 to 10,000 μm². The electrical currentapplied to the resistor in the production of the protective bubble issufficient to raise the temperature of resistor such that a protectivebubble of desired volume is produced, where the temperature of theresistor varies depending on the nature of the solvent layer.

Following selective protection of the substrate surface, the selectivelyprotected surface is contacted with a reactive agent that is capable ofreacting with unprotected susceptible groups on the substrate surface.Contact may be achieved by any convenient means. Typically, the reactiveagent is introduced into the solvent layer on the surface of thesubstrate in a manner that does not disrupt the protective bubble(s).Contact is maintained for a sufficient period of time and undersufficient conditions for the reactive agent to react with substantiallyall of the unprotected susceptible moieties present on the substratesurface. This incubation period varies depending on the nature of thereaction being performed, but generally lasts for a period of timeranging from about 5 sec to 10 min, usually from about 10 sec to 5 minand more usually from about 10 sec to 1 min. Contact of the selectivelyprotected substrate surface with the reactive agent results in thesurface modification of a portion of, but not all of, the substratesurface. In other words, the portion of the substrate surface notselectively protected by the protective bubble(s) is chemicallymodified, e.g. by the addition of a monomeric residue or by the removalof a protective group depending on whether the reactive agent is anactivated monomer or a deblocking agent, following contact with thereactive agent.

Following contact of the substrate layer with the reactive agent and thepassage of a sufficient period of time for all potential reactionsbetween the reactive agent and unprotected susceptible moieties presenton the substrate surface to occur, any remaining reactive agent (i.e.un-reactive reactive agent) is removed from the substrate surface.Removal of the un-reacted agent from substrate surface may beaccomplished using any convenient methodology. Thus, one may use washingprotocols to remove the un-reacted reactive agent. Washing protocols mayinvolve replacing the solvent layer present on the substrate surfacewith a fresh solvent layer, flooding the substrate surface with freshsolvent so to displace essentially all of the prior solvent, and thelike. During or after the washing procedure, depending on the particularprotocol employed, the selective activated resistors are deactivated. Assuch, following the washing procedure, one obtains a substrate surfacethat has been selectively modified, e.g. protective groups removed orpolymeric subunit covalently attached, in those areas that were notprotected by the protective bubbles.

Following washing, the entire substrate surface is contacted with thenext reactive agent in the polymeric synthesis protocol, e.g. anactivated monomer such as a nucleotide where the initial reactive agentcontacted with the selectively protected substrate surface is adeblocking agent; or a deblocking agent such as dichloroacetic acid ortrichloroacetic acid where the initial reactive agent is an activatednucleotide. Contact between this second reactive agent and the substratesurface is maintained for a period of time sufficient for any potentialreactions to occur between this second reactive agent and susceptiblemoieties on the substrate surface. As in the first step, un-reactedreactive agent is then removed from the substrate surface following thisincubation step, e.g. by washing.

By reiterating or repeating the above synthesis steps of selectiveprotection, reagent contact with the selectively protected surface,washing, and reagent contact with entire surface (which is notselectively protected), polymeric compounds are grown at variouspositions on the surface of the substrate. By controlling the nature ofthe reactive agents and the selective protection of the surface, anarray of diverse polymeric compounds may be synthesized on the substratesurface.

As mentioned above, the subject invention can be used to produce anumber of different types of arrays, including nucleic acid and peptidearrays. A preferred embodiment of the subject invention is the use ofthe subject methods to produce nucleic acid arrays. Nucleic acid arraysare produced according to the subject invention by synthesizing nucleicacid polymers using conventional phosphoramidite solid phase nucleicacid synthesis chemistry where the solid support is a substrate asdescribed above and protective bubbles are employed at various stages ofthe step-wise synthesis procedure to selectively protect certain siteson the substrate surface from reaction during the synthesis protocol.Phosphoramidite based chemical synthesis of nucleic acids is well knownto those of skill in the art, being reviewed in Streyer, Biochemistry(1988) pp 123-124 and U.S. Pat. No. 4,415,732, the disclosure of thelatter being herein incorporated by reference.

To produce nucleic acid arrays according to the subject methods, asubstrate surface as described above having the appropriate surfacegroups, e.g. —OH groups, present on its surface, is obtained. See theExperimental Section infra for a representative protocol for preparingsuch a substrate. Since the synthesis protocol must be carried out underanhydrous conditions, all reactions are carried out in a non-aqueous,typically organic solvent layer on the substrate surface, where thesolvent layer is acetonitrile in many embodiments.

Next, the first residues of each nucleic acid to be synthesized on thearray are covalently attached to the substrate surface via the surfacebound —OH groups. Depending on whether the first nucleotide residue ofeach nucleic acid to be synthesized on the array is the same ordifferent, different protocols for this step may be followed. Where eachof the nucleic acids to be synthesized on the substrate surface have thesame initial nucleotide at the 3′ end, the entire surface of thesubstrate is contacted with the appropriate activated nucleoside underconditions sufficient for coupling of the activated nucleoside to thereactive groups, e.g. —OH groups, present on the substrate surface tooccur. Alternatively, where the initial residue of the various nucleicacids differs among the nucleic acids, one or more sites on thesubstrate surface are initially selectively protected with protectivebubbles, as described supra, by activating the appropriate resistors(either in the substrate or the structure separate from the substrate,as described above). Following selective protection of the surface, theappropriate activated nucleotide, e.g. A, is then contacted with thesubstrate surface. Next, different sites on the substrate surface areselectively protected, followed by contact with a different activatednucleotide, e.g. C. This process is then repeated with additionaldifferent activated nucleotides, e.g. T & G, until all of the initialnucleotides of each to be synthesized nucleic acids are deposited on thesubstrate surface. At some point during this initial deposition, usuallyfollowing deposition of all of the initial nucleotides, phosphitetriesters present on the substrate surface following coupling areconverted to phosphotriesters, e.g. by oxidation with a suitableoxidating agent, such as I₂/H₂O. In addition, unreacted hydroxyl groupsare usually (though not necessarily) capped, e.g. using any convenientcapping agent, as is known in the art.

Following covalent attachment of the initial nucleotides of each nucleicacid, the following two steps are performed: (1) selectively protectingone or more positions on the substrate surface with protective bubbles;and (2) contacting the selectively protected surface with a reactiveagent, where the reactive agent may be a deblocking agent or anactivated nucleotide. Since the protocols vary depending on whether thereactive agent contacted with the selectively protected surface is adeblocking agent or an activated nucleoside phosphoramidite, each ofthese situations is described separately below.

Where the reactive agent contacted with the selectively protectedsubstrate surface is a deblocking agent, the first step followingproduction of the substrate having the first monomeric residue of eachnucleic acid on its surface is to selectively protect one or morepositions on the substrate surface with protective bubble(s). Selectiveprotection results in a portion of the sites on the substrate surfacebeing exposed to the solvent layer and a portion of the sites on thesubstrate surface being “hidden” from the solvent layer. The selectivelyprotected substrate surface is then contacted with a deblocking ordeprotecting agent, e.g. by introducing a deblocking agent into thesolvent layer. Following introduction of the deblocking agent, thesubstrate surface is incubated for a sufficient period of time underappropriate conditions for all available protecting groups, e.g. allprotecting groups not protected, i.e. underneath, a protective bubble,to be cleaved from the nucleotides that they are protecting.

Contact of the selectively protected substrate surface with a deblockingagent results in removal of the protecting groups from those substratebound residues not underneath a protective bubble. As such, this stepresults in the deprotection of a portion of the nucleotide residues onthe substrate surface. Following deprotection, the deblocking agent isremoved from the solvent layer on the surface of the substrate, whereremoval of the deblocking agent can be accomplished using any convenientprotocol. Thus, the solvent layer may be completely removed from thesubstrate surface, and then replaced with fresh solvent layer, followingone or more washing steps. Alternatively, a sufficient amount of thesolvent layer may be replaced with new solvent in a flow throughprotocol in a manner sufficient to reduce the concentration ofdeblocking agent present above the substrate surface to essentiallyzero, e.g. by flushing the substrate surface with fresh solvent. Eitherduring or after this washing step, the protective bubbles are removed,e.g. by deactivating the selectively activated resistors on either thesubstrate or the structure separate from the substrate, depending onwhich embodiment of the invention is being employed.

Removal of the deblocking agent and protective bubbles results in asubstrate surface in which a portion of the bound nucleotide residuesare deprotected and a portion of the bound nucleotide residues areprotected. In others words, removal of the protective bubbles anddeblocking agent results in the production of an array of nucleotideresidues stably associated with the substrate surface, where a portionof the nucleotide residues on the array surface have —OH groupsavailable for reaction with an activated nucleotide.

The next step in the subject methods is to contact this selectivelydeprotected array of surface bound nucleotides with an activatednucleotide under conditions sufficient for coupling between theactivated nucleotide and the deprotected surface bound nucleotide tooccur. Contact of the selectively protected surface with the activatednucleotide may be accomplished using any convenient protocol, e.g. bycontacting the surface with a solution of the activated monomer;introducing the activated monomer into a solvent layer already presenton the substrate surface; and the like. Contact of the selectivelyprotected surface with the activated nucleotide is maintained for asufficient period of time for coupling to occur. Coupling results in theproduction of a substrate having a surface in which a portion of thesurface bound nulceic acids have been elongated by one nucleotideresidue, while the remainder have not. Next, unbound activatednucleotide is removed from the surface, e.g. by washing. As is known inthe art, at some convenient point during the above steps, e.g. aftereach coupling or after all of the couplings of a given layer, thephosphite triesters that result from coupling are oxidized tophosphotriesters. The only limitation on this oxidization step is thatit generally occurs prior to the addition of the next nucleotide residueon a growing chain.

The above steps of: (a) selective protection; (b) contact of theselectively protected surface with a deblocking agent; and (c) contactof the entire surface with an activated nucleotide are repeated withadditional nucleotides until each of the desired nucleic acids on thesubstrate surface are produced. By choosing which sites are protected ateach selective protection step as well as which activated nucleotides,e.g. A, G, C & T, are contacted with the entire substrate surface, anarray having polymers of desire sequence and spatial location is readilyachieved.

The above protocol varies somewhat where the reactive agent that iscontacted with the selectively protected surface is the activatednucleotide. In this embodiment, the first step in the synthesis of thenucleic acids, following stable attachment of the terminal nucleotidesto the substrate surface, is to deblock all of the nucleotides on thesubstrate surface, e.g. to contact the entire surface with a deblockingagent. Following contact of the entire surface with a deblocking agent,the surface is selectively protected as described above, which resultsin a portion of the deblocked substrate bound nucleotides being “hidden”underneath protective bubbles. The selectively protected substratesurface is then contacted with an activated nucleotide under couplingconditions, whereby elongation occurs at those sites on the substratesurface not under a protective bubble. Following elongation, remainingactivated nucleotide is removed e.g. by washing. The surface is thenselectively protected at different locations, where at least a portionof the sites initially protected remain unprotected by a protectivebubble, and all of the unprotected sites in the first activatednucleotide contact step are generally (though not necessarily) protected(reaction should not occur at these sites since they are occupied by thefirst activated monomer which is protected). This second selectivelyprotected substrate surface is contacted with a second activated monomerto elongate the nucleic acid present at the unprotected sites. Byreiterating these steps with the remaining activated monomers, each ofthe nucleic acids on the array is elongated by one residue. Thus, thisprotocol is analogous to the first protocol, with the only differencebeing that the activated monomer is contacted with the selectivelyprotected surface and the deblocking agent is contacted with the entiresurface. As with the first protocol, by tailoring the selectiveprotection and the sequence of activated nucleotide contact, one canobtain a nucleic acid array of any desired sequence and spatialcharacteristics.

As mentioned above, the subject methods (particularly of the secondembodiment) are also amenable to the production of peptide arrays, i.e.arrays of polymeric agents which are characterized by the presence ofpeptide bonds. Any convenient solid phase peptide synthesis protocol mayadapted to practice the subject methods, where the resistor comprisingsubstrates of the subject methods are employed as the solid phase.Jones, The Chemical Synthesis of Peptides (Oxford UniversityPress)(1993) provides a review of solid phase peptide synthesis. Thepreparation of peptides according to the subject methods is analogous tothe preparation of nucleic acids described above. Thus, the reactiveagent contacted with the selectively protected surface may be adeprotecting agent or an α-amino protected amino acid. Where thereactive agent contacted with the selectively protected surface is adeblocking or deprotecting agent, the method further includes contactingthe entire surface of the substrate with an α-amino protected aminoacid. Conversely, where the reactive agent that is contacted with theselectively protected surface is an α-amino protected amino acid, thedeblocking agent is contacted with the entire surface. As with thenucleic acid synthesis, by appropriate selection of which sites toselectively protect at a give time and the order of contact of theα-amino protected amino acids with the substrate surface, a peptidearray of any desired spatial and sequence characteristics may beobtained.

The subject methods result in the production of arrays of polymericbinding agents, e.g. nucleic acid arrays, peptide arrays, etc. Thedensity and overall number of the polymeric binding agents on thesubstrate surface may vary greatly. Because of the methods by which thesubject arrays are produced, the length of each polymer on the substratesurface of the arrays generally does not exceed about 50 monomericunits, usually does not exceed about 35 monomeric units and more usuallydoes not exceed about 25 monomeric units. In those arrays producedaccording to the first embodiment of the subject invention, describedabove, a plurality of resistors are associated with the surface of thesubstrate to which the polymeric binding agents are stably attached.

The subject arrays find use in a variety applications, where suchapplications are generally analyte detection applications in which thepresence of a particular in a given sample is detected at leastqualitatively, if not quantitatively. Protocols for carrying out suchassays are well known to those of skill in the art and need not bedescribed in great detail here. Generally, the sample suspected ofcomprising the analyte of interest is contacted with an array producedaccording to the subject methods under conditions sufficient for theanalyte to bind to its respective binding pair member that is present onthe array. Thus, if the analyte of interest is present in the sample, itbinds to the array at the site of its complementary binding member and acomplex is formed on the array surface. The presence of this bindingcomplex on the array surface is then detected, e.g. through use of asignal production system, e.g. an isotopic or fluorescent label presenton the analyte, etc. The presence of the analyte in the sample is thendeduced from the detection of binding complexes on the substratesurface.

Specific analyte detection applications of interest includehybridization assays in which the nucleic acid arrays of the subjectinvention are employed. In these assays, a sample of target nucleicacids is first prepared, where preparation may include labeling of thetarget nucleic acids with a label, e.g. a member of signal producingsystem. Following sample preparation, the sample is contacted with thearray under hybridization conditions, whereby complexes are formedbetween target nucleic acids that are complementary to probe sequencesattached to the array surface. The presence of hybridized complexes isthen detected. Specific hybridization assays of interest which may bepracticed using the subject arrays include: gene discovery assays,differential gene expression analysis assays; nucleic acid sequencingassays, and the like.

Where the subject arrays are arrays having a plurality of resistorsincorporated into the substrate, e.g. such as those produced accordingto the first embodiment of the subject invention, assays can beperformed in which one or more of the resistors is selectively activatedto modulate the temperature of the reaction solution above the substratesurface, e.g. during hybridization or detection. For example, theresistors could be heated to different temperatures to get the desiredstringency for each probe type during a nucleic acid hybridizationreaction. Alternatively, for polymorphism analysis, melt-off experimentsmay be performed. In such experiments, the hybridization is allowed toproceed and then the array is scanned wet. Then each resistor is slowlyheated while the array is rescanned. As the probe/target pairs areheated, they will separate and the signal at each feature will drop. Thesignal is plotted relative to temperature (or voltage applied to theresistor). The point of inflection on the curve is called T_(m) or themelting point of the probe/target hybridization. At this point, half ofthe probe/target pairs have separated. T_(m) gives a measure of thebinding strength of the hybridization. Exact compliments have higherT_(m)'s than pairs with mismatches such as single base changes,additions or deletions.

Also provided by the subject invention are kits for use in producing thesubject arrays. Kits for producing the subject arrays generally includeat least a substrate having a plurality of individually activatableresistors associated with a surface thereof. The kits may furtherinclude various reagents that are employed in the polymeric synthesisprotocol, e.g. deblocking agent, monomers, e.g. DMT protected nucleotidephosphoramidites, activating agents, solvents, oxidizing agents, and thelike. In addition, the kits typically further include instructions forhow to synthesize a polymeric array according to the subject methods,where these instructions are generally present on at least one of apackage insert and the package of the kit.

Finally, kits for use in analyte detection assays are provided. Thesubject kits at least include the arrays of the subject invention. Thekits may further include one or more additional components necessary forcarrying out the analyte detection assay, such as sample preparationreagents, buffers, labels, and the like. In addition, the kits typicallyfurther include instructions for how practice the subject analytedetection methods according to the subject invention, where theseinstructions are generally present on at least one of a package insertand the package of the kit.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Preparation of a Resistor Substrate for Use in the Synthesis ofNucleic Acid Arrays

A resistor substrate is prepared according to the method described inU.S. Pat. No. 4,809,428, the disclosure of which is herein incorporatedby reference, with the only difference being that the barrier layer isnot applied. Instead another coating of SiO₂ is applied in thicknessranging from about 0.25-1 micron. The surface of then functionalizedaccording to the process disclosed in U.S. patent application Ser. No.09/145,015, filed Sep. 1, 1998, the disclosure of which is hereinincorporated by reference, to provide for surface —OH groups.

II. Production of Nucleic Acid Arrays.

Nucleic acid arrays are synthesized on the functionalized surface ofresistor substrates, as described above, according to the followingmethods.

A. Protecting at the Phosphoramidite Addition Step.

The substrate is built as described in Section I above through theaddition of the two passivation layers, where the SiO₂ layer is addedonly in the region of the resistors. As such, the addition of the linkerchemistry only occurs on the SiO₂ layer. The linker chemistry does nothave any protecting group and ends in a hydroxyl, thereby providing therequisite surface —OH moieties required for synthesis of the nucleicacids.

The surface is flooded in acetonitrile. Bubbles are formed over thefeatures that will not couple the first phosphoramidite. Theacetonitrile is exchanged with acetonitrile that contains the firstphosphoramidite. The coupling reaction occurs. The phosphoramiditesolution is removed by exchange with neat acetonitrile. The resistorsare turned off. Any remaining bubbles are washed off by the cappingsolutions that flood the surface. The process is repeated for the threeremaining phosphoramidites such that all of the sites have a first basecoupled. After the last coupling, the surface is flooded with theoxidizing reagents (capping if employed) and then the deblockingreagents. The process is repeated for the second round of baseadditions.

B. Protecting at the Deblock Step.

In this embodiment, the surface of the resistor substrate as describe inSection I above is modified such that the surface linker includes aprotecting group, such as dimethyloxytrityl. It is assumed that theactive surface covers the entire surface, the area over the resistorsand the area between the resistors. The first step is to inactivate thearea between the resistors. The surface is flooded with dichloromethane.Bubbles are formed at all of the resistor locations. The solvent isexchanged with 3% dichloroacetic acid or trichloroacetic acid indichloromethane to deblock the non-resistor surfaces. The solvent isexchanged with dichloromethane and the resistors are turned off. Thesurface is flooded with the capping reagents to cap all the hydroxylsbetween the features. The synthesis process can now start. The surfaceis again flooded with dichloromethane. Bubbles are formed at thoseresistor locations that will not be receiving the first phosphoramidite.The solvent is exchanged with 3% trichloroacetic acid in dichloromethaneto deblock those sites that will receive the first phosphoramidite.Again the solvent is exchanged with dichloromethane and the resistorsare turned off. The dichloromethane is removed and the surface isflooded with the first phosphoramidite. It can couple only at the sitesthat were deprotected. The phosphoramidite is removed and the surface isflooded with the capping solutions. These solutions are removed and thesurface is flooded with the oxidation reagents. These reagents areremoved and the process is started again. The next set of features isdeprotected and the second phosphoramidite is added. Normally, theprocess must be repeated four times to get a single base addition ateach location. The process is repeated until oligos of the desiredlength are built at each feature.

The resultant nucleic acid arrays find use in variety of hybridizationapplications, as describe supra.

It is evident from the above results and discussion that a simple andefficient way to prepare polymeric arrays is provided. Because thesubstrate is selectively protected by protective bubbles at appropriatestages during the polymeric synthesis process, all of the steps in thesynthesis process are alignment independent, which is a distinctadvantage over prior art methods, particularly those in which largenumbers of distinct polymers are to be positioned on the substratesurface. Furthermore, the subject methods are readily adaptable toautomated systems. As such, the subject invention is a significantcontribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method for modifying a portion of a substrate surface, said methodcomprising: (a) producing a solvent layer on said substrate surface; (b)selectively protecting at least one site on said substrate surface witha protective bubble; and (c) contacting said selectively protectedsubstrate surface with a reactive agent under conditions sufficient forsaid reactive agent to react with unprotected susceptible moietiespresent said substrate surface; whereby a portion of said substratesurface is modified.
 2. The method according to claim 1, wherein saidmethod comprises reiterating steps (b) and (c) at least one additionaltime.
 3. The method according to claim 1, wherein said method furthercomprises removing unreacted reactive agent from said substrate surfacefollowing said contact step.
 4. The method according to claim 1, whereinsaid protective bubble is produced by a bubble producing means.
 5. Themethod according to claim 4, wherein said bubble producing means is aheating means.
 6. The method according to claim 4, wherein said bubbleproducing means is a component of said substrate.
 7. The methodaccording to claim 4, wherein said bubble producing means is present ona structure separate from said substrate.
 8. A method for synthesizing aplurality of polymers on a substrate surface, said method comprising:(a) producing a solvent layer on said substrate surface, where saidsubstrate surface has a plurality of individually activatable resistorsassociated with it; (b) performing at least two iterations of thefollowing steps: (1) selectively protecting at least one site on saidsubstrate surface with a protective bubble by selective activation ofsaid resistors; (2) contacting said selectively protected substratesurface with a reactive agent under conditions sufficient for saidreactive agent to react with unprotected susceptible moieties present onsaid substrate surface; and (3) removing unreacted reactive agent fromsaid substrate surface; whereby a plurality of polymers are produced onsaid substrate surface.
 9. The method according to claim 8, wherein saidreactive agent is a deblocking agent.
 10. The method according to claim8, wherein said reactive agent is an activated monomer.
 11. The methodaccording to claim 8, wherein said polymers are nucleic acids.
 12. Amethod for synthesizing a plurality of polymers on a substrate surface,said method comprising: (a) producing a solvent layer on said substratesurface; (b) performing at least two iterations of the following steps:(1) selectively protecting at least one site on said substrate surfacewith a protective bubble, where said protective bubble is produced byactivation of a resistor present on a structure apart from saidsubstrate; (2) contacting said selectively protected substrate surfacewith a reactive agent under conditions sufficient for said reactiveagent to react with unprotected susceptible moieties present on saidsubstrate surface; and (3) removing unreacted reactive agent from saidsubstrate surface; whereby a plurality of polymers are produced on saidsubstrate surface.
 13. The method according to claim 12, wherein saidreactive agent is a deblocking agent.
 14. The method according to claim12, wherein said reactive agent is an activated monomer.
 15. The methodaccording to claim 12, wherein said polymers are nucleic acids.
 16. Themethod according to claim 12, wherein said polymers are peptides.
 17. Asubstrate produced according to the method of claim
 1. 18. A polymericarray comprising: a plurality of distinct polymers stably associatedwith the surface of a substrate, wherein said substrate comprises aplurality of individually activatable resistors associated with saidsurface and at least one of said polymers is associated with at leastone of said resistors.
 19. The polymeric array according to claim 18,wherein said plurality of resistors are beneath said surface of saidsubstrate.
 20. A nucleic acid array comprising: a plurality of nucleicacid spots stably associated with the surface of a substrate, whereinsaid substrate comprises a plurality of individually activatableresistors beneath said surface and at least one of said nucleic acidspots is associated with at least one of said resistors.
 21. A kit forproducing a polymeric array, said kit comprising: a substrate having aplurality of activatable resistors; and a deblocking agent.
 22. The kitaccording to claim 21, wherein said kit further comprises a solvent. 23.The kit according to claim 21, wherein said kit further comprisesmonomeric reagents.
 24. A method of detecting the presence of an analytein a sample, said method comprising: contacting (a) a polymeric arrayhaving a plurality of distinct polymers stably associated with thesurface of a substrate, wherein said substrate comprises a plurality ofindividually activatable resistors associated with said surface, with(b) a sample suspected of comprising said analyte under conditionssufficient for binding of said analyte to a complementary polymer onsaid array to occur; and detecting the presence of binding complexes onthe surface of the said array; whereby the presence of said analyte insaid sample is detected.
 25. The method according to claim 24, whereinsaid polymer is a nucleic acid.
 26. The method according to claim 25,wherein said analyte is a nucleic acid and said binding is byhybridization.
 27. The method according to claim 24, wherein said methodfurther comprises activating at least one of said resistors during atleast one of said contacting and detecting steps.